The applicant's long-term aims are to continue his studies on excitation contraction coupling in the heart. The specific aims are to study rabbit ventricular cells and 1. Investigate the dependence of spark probability (Ps) on [Ca]o 2. Investigate the dependence of spark probability on [Ca]o in the absence of Na gradient; 3. Investigate the fluorescence produced by Ca current and its relationship to (Ps) and, 4. Investigate the relationship between spark sites and structural morphology. The application is aimed at adducing evidence that clusters of L-type Ca channels are required to trigger sparks at high probability during a Ca transient. It is argued that spark probability is steeply dependent upon the size of the clusters of L-type Ca channels that trigger sparks. Since L-type Ca channels are reduced in diseased heart this phenomenon could explain the extinction of Ca transients in disease and as such establishes an aspect of the health relatedness of this project. Experiments are designed to show that a) the recruitment of spark sites will depend on [Ca]o and that sites triggered by smaller clusters of L-type channels will appear at lower [Ca]o. b) Sparks sites triggered by larger clusters of L-type Ca channels should show a steeper dependence of Ps on [Ca]o. These experiments will be repeated on cells in the absence of a Na gradient to establish the effect of the Na gradient on Ps. The experimental design includes a demonstration that sparks exhibit a lower Ps in the absence of a Na gradient when the SR content is matched to its value in the presence of a Na gradient. Finally the binomial distribution will be used to test the hypothesis that with the SR Ca content matched more L-type Ca channels are available to gate RyRs in the presence of a Na gradient than in its absence. As a corollary the hypothesis that not all open L-type Ca channels gate RyRs will be tested, c) The probability of spark production (Ps) is expected to correlate with the intensity of fluorescent lines that is attributable to Ca passing through clusters of L-type Ca channels. The greater the intensity of these lines, the larger the cluster of L-type Ca channels and hence the greater Ps. Finally, d) the relationship between sparks sites and local cell morphology will be established by measuring sparks and then investigating the distribution of RyRs and DHPRs in the same cell. Methods include measuring sparks and cell morphology with a combination of antibodies and mathematical deconvolution techniques. [unreadable] [unreadable] [unreadable]